The proposal is based on the hypothesis that the 2- and 4-hydroxylated metabolites of estrogens (2.and 4.OH.CEs) can contribute to the genesis of breast cancer by mechanisms involving oxidative DNA damage. The hypothesis is based on the following evidence. First, catecholestrogens (CEs) can be metabolized to quinone estrogens (QEs), that can form depurinating adducts, and can also generate reactive oxygen species (ROS) during redox cycling between CEs and QEs. Second, forms of P450 that can catalyze NADPH- dependent.2.and 4-hydroxylation of estrogens (Es) are expressed in normal human mammary ductal epithelial cells that are the cells of origin of breast cancers. Implicit in the hypothesis is that Es and xenobiotics can potentiate each others actions, since forms of P450 that catalyze metabolic activation of Es also catalyze metabolic activation of environmental procarcinogens.
Three specific aims are: 1) To complete characterizing forms of P450 expressed in human ductal epithelial cells that can catalyze CE formation by NADPH-and organic hydroperoxide-dependent mechanisms, respectively. The major remaining task in this regard is identifying form(s) of P450 that mediate organic hydroperoxide-dependent pathway of 2- and 4-OH-CEs formation . This is required since this pathway is postulated to e a major source of excess QEs and ROS. Identification will be accomplished using the strategy of RACE (rapid amplification of cDNA ends), using primers based on conserved sequences in the P450 superfamily; 2) To identify additional gene products and markers of cell damage for monitoring levels of oxidative stress in human breast parenchyma in histoculture system to be established under Specific Aim 3: a) three phase II enzymes that inactivate CEs and QEs, and that are inducible by electrophiles; b) Ref-1, a redox-responsive protein that also functions to repair oxidative DNA damage and; c) 4-hydroxynonenal, a major toxic reactive aldehyde scission product of lipid peroxidation; 3) To characterize a histoculture system of human breast parenchyma appropriate for critical testing of the CE/OE hypothesis. Human breast parenchyma, maintained on GelFoam, will be characterized with respect to: a) expression of enzymes mediating CE formation via NADPH-and OHP-dependent mechanisms; b) expression of putative markers of oxidative stress, identified under Specific Aim 2, and; c) putative markers of oxidative damage to DNA or adducts of QEs. Once the system is characterized the histoculture system will be used to test the hypothesis by determining the effect on these markers of adding Es to the histoculture, in the absence or presence of other potential sources of ROS.
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